Viruses causing morbidity and mortality in humans frequently subvert the development of an effective host immune response, which results in unrestricted virus multiplication and associated pathological manifestations. Infection of the mouse with the prototypic arenavirus LCMV provides us with a superb experimental system for the investigation of virus-host interactions contributing to both host's control of virus multiplication and viral evasion from the host antiviral response. Moreover, the significance of arenaviruses in human health and biodefense readiness, together with the limited existing armamentarium to treat arenavirus infections, highlight the importance of developing novel countermeasures to combat arenavirus infections. Antiviral therapies have been primarily focused on targeting the activity of viral gene products, an approach often compromised by the rapid selection of inhibitor-escape viral mutants. Viruses utilize and manipulate host cell factors for their multiplication and to modulate host immune responses towards favoring their survival. These host factors represent attractive and underutilized targets for antiviral therapy. Current major approaches to uncover these host factors do not account for dynamic changes in protein activity during infection. Activity-based protein profiling (ABPP) is a novel approach that permits to monitor the effects of viral infections on the functional state of the host proteome to identify novel virus-host protein interactions that affect virus propagation and pathogenesis. This exploratory R21 application will use ABPP to identify and quantify changing activity during acute and persistent infection of mice with LCMV of Serine Hydrolases (SHs), one of the largest and most diverse enzyme classes known to play important roles in many physiological processes including viral infection. For this we will complete the following specific aims: Aim 1. Characterize global spleenic SHs activity in mice during acute and persistent LCMV infection. We will use ABPP to identify changing SH activities in spleen during acute and persistent LCMV infection of the mouse. These studies will help us to begin to decipher the role of distinct SHs during viral infection. Aim 2. Determine the cellular distribution of spleenic SHs activities that re altered during acute and persistent LCMV infection. We will employ a cre-expressing recombinant LCMV to infect the mT/mG reporter mouse, which will facilitate the detection and separation of infected (GFP+) and non-infected (RFP+) cells within distinct purified immune cell populations from LCMV-infected mice. SH activities in infected and non-infected cells within purified cellular populations will be characterized by gel and mass spectrometry-based approaches as in Aim 1. Selected identified SHs will be functionally characterized regarding their roles in the regulation of the host response to LCMV infection, and specific steps of the LCMV life cycle. Results from these studies will help us to assess the biological implications of the observed changes.